For the treatment of cancer, protein therapeutics offer important advantages over conventional chemotherapy and radiation, such as high target specificity and a wide target repertoire. Unfortunately, the clinical application of protein drugs s hindered by a common set of drug delivery challenges. Proteins are degraded in the blood, deposit poorly in tumors, and are unable to cross cell membranes and access intracellular targets. It is the objective of this proposal to develop a biocompatible multifunctional polymeric delivery platform for protein drugs that facilitates (1) circulation stability, (2) tumor targeting and (3) intracellular delivery. A cutting-edge feature of the proposed polymer design is pH-dependent membrane-destabilizing activity, which allows proteins to escape acidic endosomes and access the cell cytosol. This modular drug delivery system also incorporates powerful tumor-specific antibodies and reducible disulfide groups to facilitate protein conjugation and release in the cell cytoplasm. The proposal will develop two closely related pro-apoptotic proteins, the peptide BIM and the protein BINDI engineered in the Baker lab at UW to antagonize an oncogenic Epstein-Barr virus (EBV) protein, BHRF1, with unmatched binding affinity (< 0.1 nM) and specificity. Effective therapeutic delivery or BINDI will be validated in a murine xenograft model of EBV-positive B-cell lymphoma. Furthermore, potential synergism with the chemotherapeutic agents cyclophosphamide (CY) and bortezomib will be evaluated. To achieve these objectives, three Specific Aims have been defined. In Aim 1, reversible addition fragmentation (RAFT) polymerization will be employed to synthesize diblock copolymer micelle carriers for antibody-targeted intracellular protein delivery. The carriers will be optimized for micelle size using dynamic light scattering (DLS) and pH-responsive membrane-destabilizing activity using a well-established red blood cell hemolysis assay. In Aim 2, antibody-polymer-protein conjugates will be optimized for intracellular BIM/BINDI delivery and apoptotic activity in cancer cell cancer cell lines. In Aim 3, the conjugates will be optimized in a murine xenograft model of B-cell lymphoma for (1) low toxicity in a multidose toxicity experiment, (2) tumor targeting in a pharmacokinetic/biodistribution study using fluorescently labeled protein, and (3) intratumoral apoptotic activity using a bioluminescent caspase substrate. Lastly, the optimized antibody- polymer-protein conjugate will be tested for inhibition of tumor growth and prolonged animal survival. Completion of this project will demonstrate the clinical utility of an innovative family of pH-responsive polymers for the delivery of protein cancer therapeutics. Furthermore, it will combine the Baker lab's designer proteins, the Stayton lab's drug delivery systems, the Hockenbery lab's cellular biology and in vivo imaging expertise, and the Press lab's clinical development capabilities at the Fred Hutchinson Cancer Research Center (FHCRC), in order to position an innovative and widely applicable technology for rapid clinical translation and human impact.